Complex II enzymes are integral-membrane heterotetramers with two spatially separated active sites that couple the oxidoreduction of succinate and fumarate to the oxidoreduction of quinone and quinol. In humans, Complex II is a mitochondrial enzyme that contributes to energy generation by participating in both the TCA cycle (via succinate oxidation) and oxidative phosphorylation (via quinone reduction), and its mutation is associated with tumor formation and neurodegeneration. The long-term objective of this research program is to reveal mechanisms of catalysis, assembly, and physiological function. In the proposed research, we will identify roles of Complex II assembly factors, reveal how Complex II contributes to in vivo signaling, and demonstrate how disease-associated mutations alter Complex II function. Aim 1: Until very recently, Complex II assembly was thought to proceed autocatalytically, but two assembly factors have now been identified in humans. Both are proposed to assist in cofactor insertion, but the published evidence supporting this is tenuous and there is no consensus mechanism of assembly. Understanding the roles of these assembly factors will provide insight into fundamental mechanisms of assembly of this essential protein complex. We will use Escherichia coli Complex II to reveal the role of each assembly factor, identify if they work directly or indirectly, and identify whether thre are additional assembly factors using gene deletion and complementation, mutagenesis, in vivo site-specific cross-linking, binding assays, and crystallography. Aim 2: In both humans and bacteria, Complex II activity is required for functions outside of bioenergetics. For example, in E coli, assembly of the flagellum and the chemotactic response to fumarate requires activity of the Complex II homolog QFR. Since future antimicrobials could be directed toward disruption of bacterial motility, it will be important to reveal how QFR supports flagellar assembly. We will combine site-specific cross- linking, binding assays, motility assays, crystallography, and computational modeling to identify how QFR influences the conformation of the flagellar torque ring to promote assembly and influence chemotaxis. Aim 3: Mutations in the genes encoding human Complex II are associated with pleiotropic clinical presentations. Cellular changes associated with these mutations have been reported in the literature, but almost nothing is known about the biochemical consequences of these mutations on enzyme activity. Identifying the biochemical changes associated with Complex II mutation is important for developing future therapies for mitochondrial disease. We will therefore develop an expression system for human Complex II and assess the consequences of disease-associated mutations on enzyme assembly, kinetics, and structure.